The cells at the island borders and in the three-dimensional ropes were positive for VE-Cadherin, c-kit CD41 and CD235a (Fig. 1b,c), which have been identified in mouse as markers of early yolk sac myelogenesis before definitive hematopoiesis or the establishment of embryonic circulation (<E8.5) 19. This immuno-histochemical panel is consistent with these cells representing the in vitro counterparts of early haemogenic endothelia 20. Based on this pattern of marker expression in the cystic EBs, we refer to them hereafter as Yolk Sac-EBs (YS-EBs). Large domains of the YS-EBs were positive for the master myeloid transcription factor PU.1 (Fig. 1d), which is necessary for microglial differentiation and maintenance 21,22 and can induce macrophages from fibroblasts 23. Under phase-contrast microscopy, clusters of cells were seen to delaminate towards the lumen of the cysts (Fig. 1e) as well as directly into the outer medium or on the plastic culture surface.
